Role of Interleukin 6 in Acute Pancreatitis: A Possible Marker for Disease Prognosis

Acute pancreatitis (AP) is a significant cause of morbidity, even in children, and is frequently associated with systemic manifestations. There are many cytokines involved in the inflammatory response characteristic of this disease. Interleukin 6 (IL-6) is one of the most important cytokines involved in AP, beginning from cellular injury and continuing to the systemic inflammatory response and distant organ involvement. IL-6 is a multifunctional cytokine that regulates acute-phase response and inflammation. It is produced by various cells and exerts its biological role on many cells through its high-affinity complex receptor. IL-6 has been investigated as a predicting maker for severe forms of AP. Many studies have validated the use of IL-6 serum levels in the first 48 h as a reliable marker for severe evolution and multisystemic involvement. Still, it has not been used in daily practice until now. This review discusses the main binding mechanisms by which IL-6 triggers cellular response and the AP pathogenetic mechanisms in which IL-6 is involved. We then emphasize the promising role of IL-6 as a prognostic marker, which could be added as a routine marker at admission in children with AP.


Introduction
Acute pancreatitis (AP) is a rare cause of abdominal pain with the possibility of severe evolution.In recent years, AP incidence has increased in adults and children [1,2].
The AP episodes are divided based on severity according to the revised Atlanta classification, which recognizes three forms of AP: mild (without local or systemic complications), moderate (with local complications like pancreatic/peripancreatic collections and/or organ dysfunction with resolution in less than 48 h), and severe (with organ dysfunction for more than 48 h or developing multiple organ dysfunction syndrome, MODS) [3].
Acute injury to the pancreas occurs after the exposure of acinar cells to various aggressions (gallstones, infections, trauma, toxins, or metabolites [4].In daily practice, the existence of a pancreatic injury and its severity are monitored by performing specific tests (amylase and lipase).The increase in the serum level of these parameters confirms pancreatic suffering.It gives us an overview of the severity of the injury but does not provide a precise picture of the evolutive potential of the disease.For this reason, new markers that could predict the unfavorable evolution of AP early-on have been studied.Some scoring systems have been developed but are hard to apply and do not have a good predictive value [5].Interleukin 6 (IL-6) is a biological marker that has been intensively analyzed to determine the severity of pancreatic damage, with promising results.tissue repair, inflammation, apoptosis, and adipogenesis [17].The two signaling pathways for IL-6 are represented in Figure 1A.
JAK/STAT is a common signaling pathway that transduces signals from the extracellular to the intracellular (nucleus).More than 50 cytokines, growth factors, and hormones are implicated in the JAK/STAT pathway, interfering with hematopoiesis, immune response, tissue repair, inflammation, apoptosis, and adipogenesis [17].The two signaling pathways for IL-6 are represented in Figure 1A.The IL-6 signaling.The classic pathway can be activated on cells that express both IL-6R and gp130.The trans-signaling pathway occurs through a soluble form of IL-6R that binds IL-6 and forms a complex that can act on cells that lack the IL-6R but express gp130.Both pathways activate the JAK/STAT3 cascade.(B): The involvement of IL-6 in AP pathogenesis.IL-6 is released by the necrotic acinar cells, triggers the recruitment of inflammatory cells in the pancreas (neutrophils, monocytes, T lymphocytes), and activates the resident macrophages.IL-6 also determines the activation of Th1 and Th17 cells.

IL-6 in the Pathogenesis of Acute Pancreatitis
The initiating mechanism in AP is the damage to the acinar cells.Necrotic acinar cells release damage-associated molecular patterns (DAMPs) and proinflammatory cytokines represented by IL-6, tumor necrosis factor (TNF-alpha), and interleukin-8 (IL-8), as well as anti-inflammatory cytokines, including interleukin-10 (IL-10).IL-6 is a multifunctional cytokine with pro-and anti-inflammatory properties and is one of the first cytokines involved in the inflammation associated with AP [15,18].The cytokines released initiate the recruitment of phagocytes (neutrophils and macrophages) in the pancreatic tissue.IL-6 is closely involved in neutrophil migration by trans-signaling on endothelial, smooth muscle, and epithelial cells and by upregulating the expression of adhesion molecules like ICAM-1 and VCAM-1 [6,19].After the neutrophil influx, IL-6 is involved in their burst, thus participating in the proinflammatory activity mediated by elastase, reactive oxygen species (ROS), and myeloperoxidase (MPO).More importantly, IL-6 is involved in neutrophil apoptosis, participating in the prevention of excessive inflammation [6,20].IL-6 is leading to the shift from neutrophil recruitment in the pancreas to mononuclear cell recruitment and macrophage differentiation [6].Macrophage differentiation is one of the

IL-6 in the Pathogenesis of Acute Pancreatitis
The initiating mechanism in AP is the damage to the acinar cells.Necrotic acinar cells release damage-associated molecular patterns (DAMPs) and proinflammatory cytokines represented by IL-6, tumor necrosis factor (TNF-alpha), and interleukin-8 (IL-8), as well as anti-inflammatory cytokines, including interleukin-10 (IL-10).IL-6 is a multifunctional cytokine with pro-and anti-inflammatory properties and is one of the first cytokines involved in the inflammation associated with AP [15,18].The cytokines released initiate the recruitment of phagocytes (neutrophils and macrophages) in the pancreatic tissue.IL-6 is closely involved in neutrophil migration by trans-signaling on endothelial, smooth muscle, and epithelial cells and by upregulating the expression of adhesion molecules like ICAM-1 and VCAM-1 [6,19].After the neutrophil influx, IL-6 is involved in their burst, thus participating in the proinflammatory activity mediated by elastase, reactive oxygen species (ROS), and myeloperoxidase (MPO).More importantly, IL-6 is involved in neutrophil apoptosis, participating in the prevention of excessive inflammation [6,20].IL-6 is leading to the shift from neutrophil recruitment in the pancreas to mononuclear cell recruitment and macrophage differentiation [6].Macrophage differentiation is one of the most important mechanisms determining AP's severity.The imbalance between M1 (proinflammatory) and M2 (anti-inflammatory) macrophage subtypes determines increased severity through extreme inflammation [21].IL-6 plays a bivalent role: on the one hand, it is one of the cytokines secreted by M1-activated macrophages that induce a Th1-type response and inflammatory augmentation, secreting reactive oxygen species, chemokines, and cytokines; on the other hand, it can promote alternative activation of macrophages to the wound-healing phenotype [6,22].The role of IL-6 in macrophage stimulation is not limited to the pancreatic resident macrophages.IL-6 and other proinflammatory cytokines migrate to the bloodstream and, through the portal vein, reach the liver and, afterward, the lung, activating Kupffer cells and alveolar macrophages, thus increasing inflammation and participating in the systemic progression of AP [22].
Furthermore, IL-6 plays an essential role in the acquired immune mechanisms involved in AP.IL-6 promotes the differentiation of naïve CD4 T-cells, thus being involved in the imbalance of the Th1/Th2 ratio, one of the most important mechanisms involved in AP severity [23,24].IL-6 is also an important promotor of Th17 cell proliferation and IL-17 secretion, increasing inflammatory response, primarily through activating STAT3 in naïve CD4 cells [18].The activation of the JAK/STAT signaling pathway via the signal transducing gp130 leads to the expression of various critical mediators of inflammation.It has been demonstrated that the JAK/STAT pathway activation promotes the progression of acute or chronic pancreatitis and can be the trigger for pancreatic tumors [25].Another essential function of IL-6 is the stimulation of mature B cells, thus increasing levels of B-cellactivating factor (BAFF), an important marker of inflammation and severity in AP [6,26].IL-6 is not only an inflammatory cytokine but also has anti-inflammatory action on T cells, stimulating the production of IL-10 [6].The involvement of IL-6 in AP pathogenesis is shown in Figure 1B.

IL-6 as a Prognosis Marker for Acute Pancreatitis
Predicting the severity of AP episodes is one of the most critical concerns due to the high mortality and complications associated with severe, necrotic forms.There are several scoring systems developed for adults that can be useful but do not have sufficient accuracy.Another inconvenience of these scores is that they cannot be used in the pediatric population.Thus, finding a good marker with a high predictive value that is easy to use in children is desirable [27].IL-6 is one of the most studied cytokines for this purpose.Since IL-6 is released beginning with the phase of cellular injury, it is one of the first inflammatory markers to be detected in the blood.
A study conducted by Dambrauskas et al. on 108 patients with AP demonstrated the utility of IL-6 as a prognostic marker compared with other cytokines.The evaluation of these markers was made at admission and was compared to the evolution based on imaging findings on CT on days four and seven.This study analyzed the prediction value of five cytokines (IL-6, IL-8, IL-10, MIF-macrophage migration inhibitory factor, and LTB4-leukotriene B4) for the local and systemic complications and fatal outcomes.IL-6 and MIF had the best predicting value in discriminating mild from severe forms, as well as the development of systemic complications (systemic inflammatory response syndrome-SIRS and multiple organ failure-MOF) and fatal outcomes.IL-6 was superior to MIF in differentiating edematous from necrotic forms [28].
Another study conducted by Ceranic et al. showed the superiority of IL-6 compared to other cytokine and severity scores.The study included 96 patients with AP.Severity scores (BISAP and Ranson) were calculated, and IL-6, IL-8, and IL-10 were measured at admission and after 48 h.IL-6 had a higher predictive value for severity at admission but also in the 48-h follow-up compared to IL-8 and IL-10.Furthermore, compared to the Ranson score, IL-6 measured at 48 h after admission exhibited similar predictive values for severe evolution [29].
Sternby et al. investigated the evolution of seven markers (IFN-γ, IL-1β, IL-6, IL-8, IL-10, IL-12, and TNF-α) over the first 48 h from the onset of the disease.This study concentrated on comparing the mean value of these parameters at the beginning and after the first 24 h of onset to identify the best timing for their predictive value.There were 115 patients included, classified as mild (71), moderate (33), and severe (11) according to the modified Atlanta criteria.All parameters except TNF-α had significant differences between the mild and severe groups.Still, only for IL-6, the delta values (the difference between the first and second mean values) varied significantly between all three severity groups.The main finding of this study was that the mean value of each severity group differed considerably after 24 h for IL-1β, IL-6, IL-8, and IL-10, with the most evident rise for IL-6.This may be useful for the identification of the best timing for the evaluation of these prognostic markers [30].
Kolber et al. also investigated the usefulness of IL-6 as a predicting marker for severity at admission and after 24 h.They enrolled 95 patients with AP, classified according to modified Atlanta criteria: twenty-nine mild, fifty-eight moderate, and eight severe.This study did not find significant differences between IL-6 levels upon admission and after 24 h of hospitalization.Patients with SAP had significantly higher levels of IL-6 on both days compared to the moderate and mild groups.Moreover, higher levels of IL-6 were observed in patients who developed necrosis.The level measured on the second day correlated with hospital length.There was no significant difference between the prediction accuracy of IL-6 compared with multi-variable scores (BISAP, Ranson's, or APACHE II).This is a very important finding, given that these scores are sometimes hard to determine or cannot be applied, especially if we refer to the pediatric population [31].
The study conducted by Jain et al. demonstrated the utility of IL-6 in association with SIRS for increased precision in detecting severe cases.The study was conducted on 115 patients with mild, moderate, and severe AP.SIRS was diagnosed in the first 72 h of the onset of the disease, and additionally, IL-6, TNF-α, IL-10, MCP-1, GM-CSF, and IL-1β were measured.The study revealed that the combination of SIRS at admission and IL-6 >160 pg/mL had a significantly higher PPV than SIRS alone (85% vs. 56%) and a specificity of 95%.From the other cytokines, only MPC-1 showed good results in differentiating mild from severe forms, but none of them were able to increase the sensitivity in association with SIRS.The association of IL-6 measured at admission and SIRS can be an easy-to-use tool for the early identification of severe forms, especially in children, where the other scores of severity cannot be applied [32].
Tian et al. also investigated the usefulness of the combined detection of four serum markers.They included 153 patients with AP, 81 mild and 72 severe forms.CRP, PCT, IL-6, and LDH levels were measured at admission.Each of them showed significantly different levels between mild and severe forms.However, combining these four parameters measured at admission, the sensitivity and specificity for the early detection of severe forms were significantly increased.This combination could be a simple and cost-efficient test for early severity stratification [33].
Table 1.Summarization of the evidence regarding the importance of IL-6 as a predictor marker for AP.

Authors Year Description Results
Inagaki et al. [34] 1997 12 patients: 7 with mild pancreatitis, 5 with severe pancreatitis IL-6 measured 5, 24, 72, and 120 h after admission There is a significant difference in IL-6 levels between mild and severe pancreatitis at all moments measured.The peak of IL-6 in the severe group was observed at 5 h after admission, 58.9 ± 18.8 pg/mL.Although there are other inflammatory markers, like CRP, that are more available and already used for severity prognosis in AP, many studies show a superior predictive value of IL-6, especially in the very early stages [43,53].It was observed that CRP peaks at 72 h of admission, having a delay of 24-48 h compared to IL-6, making IL-6 a more precocious marker [34,36].CRP had a relatively high value in sensitivity and accuracy after the second day of admission in predicting severe forms, necrosis, and mortality [44,54].The study conducted by De Beaux et al. showed there was no significant difference in the median serum levels of CRP on the first day of admission among patients who developed organ failure or a local pancreatic complication and those who had mild disease.By the second day of admission, the median serum level of CRP was greater in patients who developed organ failure than in those with a local pancreatic complication (p = 0.02) or with mild disease alone (p = 0.003) [48].All these data, alongside other studies showing the usefulness of the combined assessment of IL-6 and CRP, concluded that IL-6 is not a replacement for CRP but rather an earlier or a complementary marker.

Limitations and Future Directions
As shown above, numerous data support the usefulness of IL-6 as an early predictor for severity in AP.Still, it has not been introduced in clinical practice because of some limitations.Firstly, IL-6 is involved in the very first stages of AP, and it is not stored in cells, meaning that it could have a rapid increase but also a decrease in serum levels.Thus, the measurement of serum levels has to be done close to the onset of the symptoms.In clinical practice, this may prove difficult, as it is sometimes hard to determine the exact timeline.Although all studies discussed above demonstrated the utility of IL-6 as a severity predictor, the timing for measurement is not very well-established.The majority of data indicate the first 48 h after admission as the best timeframe.
Secondly, there are no clear cut-off values.Some of the studies did not evaluate this parameter, or in the studies that are present, there are extremely large variations.
Moreover, the cost and availability of cytokine measurement is another limitation for routine use in daily practice.Although in the past this was the primary limitation, currently more and more methods are widely available with moderate costs.Furthermore, a closer analysis of the other severity scores shows us that they are time-consuming and need other laboratory parameters and imaging evaluation.At the same time, IL-6 measurement is rapid and can be used in children as well as adults.
Serum IL-6 measured in the first 48 h of admission could be used in clinical practice as a single parameter or as part of a new score associated with other serum markers.A point-of-care test for IL-6 has been used in different diseases and could be used in AP for decision-making and treatment strategy.More data is needed to establish unitary cut-off values for severity stages and systemic complications.

Conclusions
Severity stratification and prediction of severe, necrotic forms of AP is essential in managing the disease due to its rapid and unpredictable evolution.A reliable serum marker or a combination of markers could be the best approach for more standardized care.IL-6 is the most promising marker, with the best results, and should enter the panel of markers analyzed in patients with AP, including children.

Figure 1 .
Figure 1.(A): The IL-6 signaling.The classic pathway can be activated on cells that express both IL-6R and gp130.The trans-signaling pathway occurs through a soluble form of IL-6R that binds IL-6 and forms a complex that can act on cells that lack the IL-6R but express gp130.Both pathways activate the JAK/STAT3 cascade.(B): The involvement of IL-6 in AP pathogenesis.IL-6 is released by the necrotic acinar cells, triggers the recruitment of inflammatory cells in the pancreas (neutrophils, monocytes, T lymphocytes), and activates the resident macrophages.IL-6 also determines the activation of Th1 and Th17 cells.

Figure 1 .
Figure 1.(A): The IL-6 signaling.The classic pathway can be activated on cells that express both IL-6R and gp130.The trans-signaling pathway occurs through a soluble form of IL-6R that binds IL-6 and forms a complex that can act on cells that lack the IL-6R but express gp130.Both pathways activate the JAK/STAT3 cascade.(B): The involvement of IL-6 in AP pathogenesis.IL-6 is released by the necrotic acinar cells, triggers the recruitment of inflammatory cells in the pancreas (neutrophils, monocytes, T lymphocytes), and activates the resident macrophages.IL-6 also determines the activation of Th1 and Th17 cells.